Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8

Abstract

Z.Kozmik and T.Czerny contributed equally to this workTranscription factors of the Pax family bind to their target genes via the paired domain which is known to be composed of two subdomains each recognizing distinct half-sites in adjacent major grooves of the DNA helix. We now demonstrate that the mammalian Pax8 gene gives rise, by alternative mRNA splicing, to a protein isoform containing an extra serine residue in the recognition α-helix 3 of the paired domain. This Pax8(S) protein does not interact with bipartite paired domain-binding sites, indicating that inactivation of the N-terminal DNA-binding motif severely restricts the sequence specificity of the paired domain. However, the Pax8(S) protein binds in vitro and in vivo to the 5aCON sequence which was previously identified as a high-affinity binding site for the Pax6(5a) splice variant carrying a 14-amino-acid insertion in the paired domain. The 5aCON sequence is shown to consist of four interdigitated 5Ј half-sites of the bipartite consensus sequence and is thus bound by four Pax8(S) molecules via the intact C-terminal DNA-binding motif of the paired domain. Together these data suggest that inactivation of the N-terminal region of the paired domain by alternative splicing is used in vivo to selectively target Pax transcription factors to gene regulatory regions containing highly specialized 5aCON-like sequences.

Growth and specification of the eye are controlled independently by Eyegone and Eyeless in Drosophila melanogaster

“…The sole PAX6 gene in humans produces two primary isoforms, PAX6(5a) and PAX6, which differ in the inclusion of 14 amino acids encoded by exon 5a in the N-terminal subdomain of the paired domain 10,[18][19][20] (Fig. 1a).…”

“…The paired domain is a bipartite DNA-binding domain of 128 amino acids, consisting of N-terminal and C-terminal subdomains, each recognizing distinct DNA sites [18][19][20] . Although the N-terminal and C-terminal subregions of the paired domain can bind DNA with high affinity when expressed separately, in the context of an intact paired domain, Pax proteins bind DNA preferentially through the N-terminal subregion.…”

“…Although the N-terminal and C-terminal subregions of the paired domain can bind DNA with high affinity when expressed separately, in the context of an intact paired domain, Pax proteins bind DNA preferentially through the N-terminal subregion. The DNA-binding potential of the C terminus is only manifest when the N-terminal activity is blocked [18][19][20] . For instance, an alternative splicing event in the sole PAX6 gene in humans and rodents creates the isoform PAX6(5a) 10,19 , which lacks the normal paired domain activity owing to the inclusion of an extra exon in the N-terminal subregion (Fig.…”

“…This isoform recognizes DNA exclusively through the C-terminal subregion of the paired domain. Likewise, alternative use of exons in the paired domains of PAX3 and PAX8 also results in the production of isoforms that use the C-terminal subdomain for DNA binding 6,[18][19][20] . The purpose of masking the C-terminal activity in the intact paired-domain proteins is unclear because the biological functions of these isoforms are unknown.…”

“…1a). Biochemical studies showed that Eyg binds through the C terminus of the paired domain to a 5aCON-like site 30 , which corresponds to the high-affinity binding site for mammalian Pax splice isoforms, such as PAX6(5a) [18][19][20] . Eyg is characterized as an eye-specifying factor [31][32][33] , and overexpression studies suggested that Eyg and Ey might substitute for each other functionally 31 .…”